Intercellular calcium waves in primary cultured rat mesenteric smooth muscle cells are mediated by connexin43

Intercellular Ca(2+) wave propagation between vascular smooth muscle cells (SMCs) is associated with the propagation of contraction along the vessel. Here, we characterize the involvement of gap junctions (GJs) in Ca(2+) wave propagation between SMCs at the cellular level. Gap junctional communication was assessed by the propagation of intercellular Ca(2+) waves and the transfer of Lucifer Yellow in A7r5 cells, primary rat mesenteric SMCs (pSMCs), and 6B5N cells, a clone of A7r5 cells expressing higher connexin43 (Cx43) to Cx40 ratio. Mechanical stimulation induced an intracellular Ca(2+) wave in pSMC and 6B5N cells that propagated to neighboring cells, whereas Ca(2+) waves in A7r5 cells failed to progress to neighboring cells. We demonstrate that Cx43 forms the functional GJs that are involved in mediating intercellular Ca(2+) waves and that co-expression of Cx40 with Cx43, depending on their expression ratio, may interfere with Cx43 GJ formation, thus altering junctional communication.     

In Situ Microscopy Analysis Reveals Local Innate Immune Response Developed around Brucella Infected Cells in Resistant and Susceptible Mice

Brucella are facultative intracellular bacteria that chronically infect humans and animals causing brucellosis. Brucella are able to invade and replicate in a broad range of cell lines in vitro, however the cells supporting bacterial growth in vivo are largely unknown. In order to identify these, we used a Brucella melitensis strain stably expressing mCherry fluorescent protein to determine the phenotype of infected cells in spleen and liver, two major sites of B. melitensis growth in mice. In both tissues, the majority of primary infected cells expressed the F4/80 myeloid marker. The peak of infection correlated with granuloma development. These structures were mainly composed of CD11b⁺ F4/80⁺ MHC-II⁺ cells expressing iNOS/NOS2 enzyme. A fraction of these cells also expressed CD11c marker and appeared similar to inflammatory dendritic cells (DCs). Analysis of genetically deficient mice revealed that differentiation of iNOS⁺ inflammatory DC, granuloma formation and control of bacterial growth were deeply affected by the absence of MyD88, IL-12p35 and IFN-γ molecules. During chronic phase of infection in susceptible mice, we identified a particular subset of DC expressing both CD11c and CD205, serving as a reservoir for the bacteria. Taken together, our results describe the cellular nature of immune effectors involved during Brucella infection and reveal a previously unappreciated role for DC subsets, both as effectors and reservoir cells, in the pathogenesis of brucellosis.     

Synthesis of new N-isobutyryl-L-cysteine/MEA conjugates: evaluation of their free radical-scavenging activities and anti-HIV properties in human macrophages

Four novel N-isobutyryl-l-cysteine/2-mercaptoethylamine (MEA, cysteamine) conjugates have been designed and synthesized. The antioxidant activities of these new series were evaluated by three different free radical scavenging methods (DPPH test, ABTS test, and deoxyribose assay) and their metal binding capacity was evaluated by the ethidium bromide fluorescence binding assay. These results were compared with those obtained with their pro-GSH acetyl analogues recently developed in our laboratory. We observed that most of these compounds exhibit free radical-scavenging activities similar to those of Trolox, but always superior than NAC. While none of these new derivatives had pro-GSH activities, they displayed anti-HIV properties in human monocyte-derived macrophages infected in vitro. The present study demonstrates that these new N-isobutyryl derivatives, which are expected to have a greater bioavailability than their acetyl analogues, may have useful applications in HIV infection in respect to their antioxidant and anti-HIV activities.     

Automated region of interest retrieval and classification using spectral analysis

Efficient use of whole slide imaging in pathology needs automated region of interest (ROI) retrieval and classification, through the use of image analysis and data sorting tools. One possible method for data sorting uses Spectral Analysis for Dimensionality Reduction. We present some interesting results in the field of histopathology and cytohematology. In histopathology, we developed a Computer-Aided Diagnosis system applied to low-resolution images representing the totality of histological breast tumour sections. The images can be digitized directly at low resolution or be obtained from sub-sampled high-resolution virtual slides. Spectral Analysis is used (1) for image segmentation (stroma, tumour epithelium), by determining a "distance" between all the images of the database, (2) for choosing representative images and characteristic patterns of each histological type in order to index them, and (3) for visualizing images or features similar to a sample provided by the pathologist. In cytohematology, we studied a blood smear virtual slide acquired through high resolution oil scanning and Spectral Analysis is used to sort selected nucleated blood cell classes so that the pathologist may easily focus on specific classes whose morphology could then be studied more carefully or which can be analyzed through complementary instruments, like Multispectral Imaging or Raman MicroSpectroscopy.     

Comparative dynamics of retrograde actin flow and focal adhesions: formation of nascent adhesions triggers transition from fast to slow flow

Dynamic actin network at the leading edge of the cell is linked to the extracellular matrix through focal adhesions (FAs), and at the same time it undergoes retrograde flow with different dynamics in two distinct zones: the lamellipodium (peripheral zone of fast flow), and the lamellum (zone of slow flow located between the lamellipodium and the cell body). Cell migration involves expansion of both the lamellipodium and the lamellum, as well as formation of new FAs, but it is largely unknown how the position of the boundary between the two flow zones is defined, and how FAs and actin flow mutually influence each other. We investigated dynamic relationship between focal adhesions and the boundary between the two flow zones in spreading cells. Nascent FAs first appeared in the lamellipodium. Within seconds after the formation of new FAs, the rate of actin flow decreased locally, and the lamellipodium/lamellum boundary advanced towards the new FAs. Blocking fast actin flow with cytochalasin D resulted in rapid dissolution of nascent FAs. In the absence of FAs (spreading on poly-L-lysine-coated surfaces) retrograde flow was uniform and the velocity transition was not observed. We conclude that formation of FAs depends on actin dynamics, and in its turn, affects the dynamics of actin flow by triggering transition from fast to slow flow. Extension of the cell edge thus proceeds through a cycle of lamellipodium protrusion, formation of new FAs, advance of the lamellum, and protrusion of the lamellipodium from the new base.     

Oncologic Trogocytosis of an Original Stromal Cells Induces Chemoresistance of Ovarian Tumours

Background

The microenvironment plays a major role in the onset and progression of metastasis. Epithelial ovarian cancer (EOC) tends to metastasize to the peritoneal cavity where interactions within the microenvironment might lead to chemoresistance. Mesothelial cells are important actors of the peritoneal homeostasis; we determined their role in the acquisition of chemoresistance of ovarian tumours.

Methodology/Principal Findings

We isolated an original type of stromal cells, referred to as “Hospicells” from ascitis of patients with ovarian carcinosis using limiting dilution. We studied their ability to confer chemoresistance through heterocellular interactions. These stromal cells displayed a new phenotype with positive immunostaining for CD9, CD10, CD29, CD146, CD166 and Multi drug resistance protein. They preferentially interacted with epithelial ovarian cancer cells. This interaction induced chemoresistance to platin and taxans with the implication of multi-drug resistance proteins. This contact enabled EOC cells to capture patches of the Hospicells membrane through oncologic trogocytosis, therefore acquiring their functional P-gp proteins and thus developing chemoresistance. Presence of Hospicells on ovarian cancer tissue micro-array from patients with neo-adjuvant chemotherapy was also significantly associated to chemoresistance.

Conclusions/Significance

This is the first report of trogocytosis occurring between a cancer cell and an original type of stromal cell. This interaction induced autonomous acquisition of chemoresistance. The presence of stromal cells within patient''s tumour might be predictive of chemoresistance. The specific interaction between cancer cells and stromal cells might be targeted during chemotherapy.     

Uterine contractions depend on KIT-positive interstitial cells in the mouse: genetic and pharmacological evidence

In the gastrointestinal tract, interstitial cells of Cajal (ICCs) generate a pacemaker activity. They produce electric slow waves that trigger and coordinate gut smooth muscle contractions. Interstitial cells of Cajal's slender shape is revealed by KIT immunostaining. Based on several features, including KIT expression and KIT dependence, ICC-like cells were identified in nongastrointestinal tissues. Here, we investigated in the mouse whether uterine contractions depend on ICC-like cells' activity. By labeling KIT-expressing cells, we found putative ICC-like cells in the uterus, observed as KIT-positive interstitial, long spindle-shaped cells with fine branched cytoplasm processes, distributed in muscular layers and in subepithelial connective tissue. We then checked the potential KIT dependence of ex vivo contractile activity of the uterus by combining genetic and pharmacological approaches, using the Kit W-v hypomorphic mutation, and imatinib as a KIT noncompetitive inhibitor. We found a significant reduction in frequency of longitudinal uterine contractions in Kit W-v/Kit W-v compared with Kit+/+ mice, whereas amplitude was unaffected. There was no difference in frequency or amplitude of circular uterine contractions between Kit W-v/Kit W-v and Kit+/+ mice. Ex vivo treatment of Kit+/+ uterine horns with imatinib resulted in a dose-dependent reduction of the frequency and amplitude of longitudinal myometrial contractions. Amplitude and frequency of circular contractions were unaffected in presence of imatinib. These concurrent results suggest that longitudinal contractions of the uterus depend on a KIT signaling pathway of ICC-like cells. The existence of ICC-like cells in the myometrium may enhance our understanding of uterine spontaneous contractile activity and suggest new approaches for treatment of uterine contractility disorders.     

Bax-induced cell death in yeast depends on mitochondrial lipid oxidation.

The oxidant function of pro-apoptotic protein Bax was investigated through heterologous expression in yeast. Direct measurements of fatty acid content show that Bax-expression induces oxidation of mitochondrial lipids. This effect is prevented by the coexpression of Bcl-xL. The oxidation actually could be followed on isolated mitochondria as respiration-induced peroxidation of polyunsaturated cis-parinaric acid and on whole cells as the increase in the amount of thiobarbituric acid-reactive products. Treatments that increase the unsaturation ratio of lipids, making them more sensitive to oxidation, increase kinetics of Bax-induced death. Conversely, inhibitors of lipid oxidation and treatments that decrease the unsaturation ratio of fatty acids decrease kinetics of Bax-induced death. Taken together, these results show that Bax-induced mitochondrial lipid oxidation is relevant to Bax-induced cell death. Conversely, lipid oxidation is poorly related to the massive Bax-induced superoxide and hydrogen peroxide accumulation, which occurs at the same time, as chemical or enzymatic scavenging of ROS does not prevent lipid oxidation nor has any effects on kinetics of Bax-induced cell death. Whatever the origin of mitochondrial lipid oxidation, these data show that it represents a major step in the cascade of events leading to Bax-induced cell death. These results are discussed in the light of the role of lipid oxidation both in mammalian apoptosis and in other forms of cell death in other organisms.     

Escherichia coli produces phosphoantigens activating human gamma delta T cells.

Human Vgamma9delta2 T lymphocytes are suggested to play an important role in the immune response to various microbial pathogens. In contrast to alphabeta T cells, gammadelta T lymphocytes recognize small, non-protein, phosphate-bearing antigens (phosphoantigens) in a major histocompatibility complex-independent manner. Four different phosphoantigens termed TUBag1 to TUBag4 with a common 3-formyl-1-butyl-pyrophosphate moiety and isopentenyl-pyrophosphate have been isolated and identified from mycobacteria. However, natural occurring gammadelta T cell ligands from other bacterial species were not characterized so far. Here, we describe the structural identification of the two compounds responsible for the gammadelta T cell-stimulating capacity of Escherichia coli as similar to the mycobacterial phosphoantigens 3-formyl-1-butyl-pyrophosphate and its M(r) 275 homologue TUBag2. In addition, E. coli phosphoantigens exert bioactivities on gammadelta T cells with similar potencies to the mycobacterial phosphoantigens at 5-15 nm concentration. Furthermore, our results clearly prove that the deoxyxylulose 5-phophate pathway (also referred to as Rohmer metabolic route of isoprenoid biosynthesis) is essential for the biosynthesis of the phosphoantigens in E. coli. Because this pathway is absent from human cells, it proves an ideal target for focusing efficiently the antimicrobial selectivity of human gammadelta T lymphocytes.