Neural Mechanisms Underlying Breathing Complexity

Breathing is maintained and controlled by a network of automatic neurons in the brainstem that generate respiratory rhythm and receive regulatory inputs. Breathing complexity therefore arises from respiratory central pattern generators modulated by peripheral and supra-spinal inputs. Very little is known on the brainstem neural substrates underlying breathing complexity in humans. We used both experimental and theoretical approaches to decipher these mechanisms in healthy humans and patients with chronic obstructive pulmonary disease (COPD). COPD is the most frequent chronic lung disease in the general population mainly due to tobacco smoke. In patients, airflow obstruction associated with hyperinflation and respiratory muscles weakness are key factors contributing to load-capacity imbalance and hence increased respiratory drive. Unexpectedly, we found that the patients breathed with a higher level of complexity during inspiration and expiration than controls. Using functional magnetic resonance imaging (fMRI), we scanned the brain of the participants to analyze the activity of two small regions involved in respiratory rhythmogenesis, the rostral ventro-lateral (VL) medulla (pre-Bötzinger complex) and the caudal VL pons (parafacial group). fMRI revealed in controls higher activity of the VL medulla suggesting active inspiration, while in patients higher activity of the VL pons suggesting active expiration. COPD patients reactivate the parafacial to sustain ventilation. These findings may be involved in the onset of respiratory failure when the neural network becomes overwhelmed by respiratory overload We show that central neural activity correlates with airflow complexity in healthy subjects and COPD patients, at rest and during inspiratory loading. We finally used a theoretical approach of respiratory rhythmogenesis that reproduces the kernel activity of neurons involved in the automatic breathing. The model reveals how a chaotic activity in neurons can contribute to chaos in airflow and reproduces key experimental fMRI findings.     

Flow Cytometric Quantification of All Phases of the Cell Cycle and Apoptosis in a Two-Color Fluorescence Plot

An optimal technology for cell cycle analysis would allow the concomitant measurement of apoptosis, G0, G1, S, G2 and M phases in combination with cell surface phenotyping. We have developed an easy method in flow cytometry allowing this discrimination in an only two-color fluorescent plot. It is based on the concomitant use of 7-amino-actinomycin D and the antibodies anti-Ki67 and anti-phospho(Ser10)-histone H3, both conjugated to Alexa Fluor®488 to discriminate G0 and M phases, respectively. The method is particularly valuable in a clinical setting as verified in our laboratory by analyzing human leukemic cells from marrow samples or after exposure to cell cycle modifiers.     

High Content Analysis of Primary Macrophages Hosting Proliferating Leishmania Amastigotes: Application to Anti-leishmanial Drug Discovery

Background/Objectives

Human leishmaniases are parasitic diseases causing severe morbidity and mortality. No vaccine is available and numerous factors limit the use of current therapies. There is thus an urgent need for innovative initiatives to identify new chemotypes displaying selective activity against intracellular Leishmania amastigotes that develop and proliferate inside macrophages, thereby causing the pathology of leishmaniasis.

Methodology/Principal Findings

We have developed a biologically sound High Content Analysis assay, based on the use of homogeneous populations of primary mouse macrophages hosting Leishmania amazonensis amastigotes. In contrast to classical promastigote-based screens, our assay more closely mimics the environment where intracellular amastigotes are growing within acidic parasitophorous vacuoles of their host cells. This multi-parametric assay provides quantitative data that accurately monitors the parasitic load of amastigotes-hosting macrophage cultures for the discovery of leishmanicidal compounds, but also their potential toxic effect on host macrophages. We validated our approach by using a small set of compounds of leishmanicidal drugs and recently published chemical entities. Based on their intramacrophagic leishmanicidal activity and their toxicity against host cells, compounds were classified as irrelevant or relevant for entering the next step in the drug discovery pipeline.

Conclusions/Significance

Our assay represents a new screening platform that overcomes several limitations in anti-leishmanial drug discovery. First, the ability to detect toxicity on primary macrophages allows for discovery of compounds able to cross the membranes of macrophage, vacuole and amastigote, thereby accelerating the hit to lead development process for compounds selectively targeting intracellular parasites. Second, our assay allows discovery of anti-leishmanials that interfere with biological functions of the macrophage required for parasite development and growth, such as organelle trafficking/acidification or production of microbicidal effectors. These data thus validate a novel phenotypic screening assay using virulent Leishmania amastigotes growing inside primary macrophage to identify new chemical entities with bona fide drug potential.     

Impact of Neutrophil-Secreted Myeloid Related Proteins 8 and 14 (MRP 8/14) on Leishmaniasis Progression

The myeloid-related proteins (MRPs) 8/14 are small proteins mainly produced by neutrophils, which have been reported to induce NO production in macrophages. On the other hand, Leishmania survives and multiplies within phagocytes by inactivating several of their microbicidal functions. Whereas MRPs are rapidly released during the innate immune response, their role in the regulation of Leishmaniasis is still unknown. In vitro experiments revealed that Leishmania infection alters MRP-induced signaling, leading to inhibition of macrophage functions (NO, TNF-α). In contrast, MRP-primed cells showed normal signaling activation and NO production in response to Leishmania infection. Using a murine air-pouch model, we observed that infection with L. major induced leukocyte recruitment and MRP secretion comparable to LPS-treated mice. Depletion of MRPs significantly reduced these inflammatory events and augmented both parasite load and footpad swelling during the first 8 weeks post-infection, as also observed in MRP KO mice. On the contrary, mouse treatment with recombinant MRPs (rMRPs) had the opposite effect. Collectively, our results suggest that rapid secretion of MRPs by neutrophils at the site of infection may protect uninfected macrophages and favor a more efficient innate inflammatory response against Leishmania infection. In summary, our study reveals the critical role played by MRPs in the regulation of Leishmania infection and how this pathogen can subvert its action.     

Frondoside A Suppressive Effects on Lung Cancer Survival,Tumor Growth,Angiogenesis, Invasion,and Metastasis

A major challenge for oncologists and pharmacologists is to develop less toxic drugs that will improve the survival of lung cancer patients. Frondoside A is a triterpenoid glycoside isolated from the sea cucumber, Cucumaria frondosa and was shown to be a highly safe compound. We investigated the impact of Frondoside A on survival, migration and invasion in vitro, and on tumor growth, metastasis and angiogenesis in vivo alone and in combination with cisplatin. Frondoside A caused concentration-dependent reduction in viability of LNM35, A549, NCI-H460-Luc2, MDA-MB-435, MCF-7, and HepG2 over 24 hours through a caspase 3/7-dependent cell death pathway. The IC50 concentrations (producing half-maximal inhibition) at 24 h were between 1.7 and 2.5 µM of Frondoside A. In addition, Frondoside A induced a time- and concentration-dependent inhibition of cell migration, invasion and angiogenesis in vitro. Frondoside A (0.01 and 1 mg/kg/day i.p. for 25 days) significantly decreased the growth, the angiogenesis and lymph node metastasis of LNM35 tumor xenografts in athymic mice, without obvious toxic side-effects. Frondoside A (0.1–0.5 µM) also significantly prevented basal and bFGF induced angiogenesis in the CAM angiogenesis assay. Moreover, Frondoside A enhanced the inhibition of lung tumor growth induced by the chemotherapeutic agent cisplatin. These findings identify Frondoside A as a promising novel therapeutic agent for lung cancer.     

Plasticity of Migrating CD1b+ and CD1b- Lymph Dendritic Cells in the Promotion of Th1, Th2 and Th17 in Response to Salmonella and Helminth Secretions

Dendritic cells (DCs) are pivotal in the development of specific T-cell responses to control pathogens, as they govern both the initiation and the polarization of adaptive immunity. To investigate the capacities of migrating DCs to respond to pathogens, we used physiologically generated lymph DCs (L-DCs). The flexible polarization of L-DCs was analysed in response to Salmonella or helminth secretions known to induce different T cell responses. Mature conventional CD1b⁺ L-DCs showed a predisposition to promote pro-inflammatory (IL-6), pro-Th1 (IL-12p40) and anti-inflammatory (IL-10) responses which were amplified by Salmonella, and limited to only IL-6 induction by helminth secretions. The other major population of L-DCs did not express the CD1b molecule and displayed phenotypic features of immaturity compared to CD1b⁺ L-DCs. Salmonella infection reduced the constitutive expression of TNF-α and IL-4 mRNA in CD1b^- L-DCs, whereas this expression was not affected by helminth secretions. The cytokine response of T cells promoted by L-DCs was analysed in T cell subsets after co-culture with Salmonella or helminth secretion-driven CD1b⁺ or CD1b^- L-DCs. T cells preferentially expressed the IL-17 gene, and to a lesser extent the IFN-γ and IL-10 genes, in response to Salmonella-driven CD1b⁺ L-DCs, whereas a preferential IL-10, IFN-γ and IL-17 gene expression was observed in response to Salmonella-driven CD1b^- L-DCs. In contrast, a predominant IL-4 and IL-13 gene expression by CD4⁺ and CD8⁺ T cells was observed after stimulation of CD1b⁺ and CD1b^- L-DCs with helminth secretions. These results show that mature conventional CD1b⁺ L-DCs maintain a flexible capacity to respond differently to pathogens, that the predisposition of CD1b^- L-DCs to promote a Th2 response can be oriented towards other Th responses, and finally that the modulation of migrating L-DCs responses is controlled more by the pathogen encountered than the L-DC subsets.     

Hematological and hemorheological Determinants of the Six-Minute Walk Test Performance in Children with Sickle Cell Anemia

The six-minute walk test is a well-established submaximal exercise reflecting the functional status and the clinical severity of sickle cell patients. The aim of the present cross-sectional study was to investigate the biological determinants of the six-minute walk test performance in children with sickle cell anemia. Hematological and hemorheological parameters, pulmonary function and the six-minute walk test performance were determined in 42 children with sickle cell anemia at steady state. The performance during the six-minute walk test was normalized for age, sex and height and expressed as percentage of the predicted six-minute walk distance. We showed that a high level of anemia, a low fetal hemoglobin expression and low red blood cell deformability were independent predictors of a low six-minute walk test performance. This study describes for the first time the impact of blood rheology in the six-minute walk test performance in children with sickle cell anemia.     

Role of Prostate Apoptosis Response 4 in Translocation of GRP78 from the Endoplasmic Reticulum to the Cell Surface of Trophoblastic Cells

Glucose-regulated protein 78 (GRP78) is an endoplasmic reticulum (ER) molecular chaperone that belongs to the heat shock protein 70 family. GRP78 is also present on the cell surface membrane of trophoblastic cells, where it is associated with invasive or fusion properties of these cells. Impaired mechanism of GRP78 relocation from ER to the cell surface was observed in preeclamptic cytotrophoblastic cells (CTB) and could take part in the pathogenesis of preeclampsia. In this study, we have investigated whether prostate apoptosis response 4 (Par-4), a protein identified as a partner of GRP78 relocation to the cell surface in prostate cancer cells, is present in trophoblastic cells and is involved in the translocation of GRP78 to the cell surface of CTB. Par-4 is indeed present in trophoblastic cells and its expression correlates with expression of membrane GRP78. Moreover, overexpression of Par-4 led to an increase of cell surface expression of GRP78 and decreased Par-4 gene expression reduced cell surface localization of GRP78 confirming a role of Par-4 in relocation of GRP78 from ER to the cell surface. Accordingly, invasive property was modified in these cells. In conclusion, we show that Par-4 is expressed in trophoblastic cells and is involved in transport of GRP78 to the cell surface and thus regulates invasive property of extravillous CTB.     

Evolution of Exchangeable Copper and Relative Exchangeable Copper through the Course of Wilson's Disease in the Long Evans Cinnamon Rat

Background

Wilson''s disease (WD) is an inherited disorder of copper metabolism leading to liver failure and/or neurological impairment. Its diagnosis often remains difficult even with genetic testing. Relative exchangeable copper (REC) has recently been described as a reliable serum diagnostic marker for WD.

Methodology/Principal Findings

The aim of this study was to validate the use of REC in the Long Evans Cinnamon (LEC) rat, an animal model for WD, and to study its relevance under different conditions in comparison with conventional markers. Two groups of LEC rats and one group of Long-Evans (LE) rats were clinically and biologically monitored from 6 to 28 weeks of age. One group of LEC rats was given copper-free food. The other groups had normal food. Blood samples were collected each month and different serum markers for WD (namely ceruloplasmin oxidase activity, exchangeable copper (CuEXC), total serum copper and REC) and acute liver failure (serum transaminases and bilirubinemia) were tested. Every LEC rat under normal food developed acute liver failure (ALF), with 40% global mortality. Serum transaminases and bilirubinemia along with total serum copper and exchangeable copper levels increased with the onset of acute liver failure. A correlation was observed between CuEXC values and the severity of ALF. Cut-off values were different between young and adult rats and evolved because of age and/or liver failure. Only REC, with values >19%, was able to discriminate LEC groups from the LE control group at every time point in the study. REC sensitivity and specificity reached 100% in adults rats.

Conclusions/Significance

REC appears to be independent of demographic or clinical data in LEC rats. It is a very simple and reliable blood test for the diagnosis of copper toxicosis owing to a lack of ATP7B function. CuEXC can be used as an accurate biomarker of copper overload.