As Toll-like receptors (TLRs) are expressed by hematopoietic stem and progenitor cells (HSPCs), they may play a role in hematopoiesis in response to pathogens during infection. We show here that ...TLR2, TLR4, and TLR9 agonists (tripalmitoyl-S-glyceryl-L-Cys-Ser-(Lys)4 Pam3CSK4, lipopolysaccharide LPS, and CpG oligodeoxynucleotide ODN) induce the in vitro differentiation of purified murine lineage negative cells (Lin(-) ) as well as HSPCs (identified as Lin(-) c-Kit(+) Sca-1(+) IL-7Rα(-) LKS cells) toward macrophages (Mph), through a myeloid differentiation factor 88 (MyD88)-dependent pathway. In order to investigate the possible direct interaction of soluble microorganism-associated molecular patterns and TLRs on HSPCs in vivo, we designed a new experimental approach: purified Lin(-) and LKS cells from bone marrow of B6Ly5.1 mice (CD45.1 alloantigen) were transplanted into TLR2(-/-) , TLR4(-/-) , or MyD88(-/-) mice (CD45.2 alloantigen), which were then injected with soluble TLR ligands (Pam3CSK4, LPS, or ODN, respectively). As recipient mouse cells do not recognize the TLR ligands injected, interference by soluble mediators secreted by recipient cells is negligible. Transplanted cells were detected in the spleen and bone marrow of recipient mice, and in response to soluble TLR ligands, cells differentiated preferentially to Mph. These results show, for the first time, that HSPCs may be directly stimulated by TLR agonists in vivo, and that the engagement of these receptors induces differentiation toward Mph. Therefore, HSPCs may sense pathogen or pathogen-derived products directly during infection, inducing a rapid generation of cells of the innate immune system.
The present study was designed to define an experimental model of hepatocellular steatosis with a fat overaccumulation profile in which the metabolic and cytotoxic/apoptotic effects could be ...separated. This was accomplished by defining the experimental conditions of lipid exposure that lead to significant intracellular fat accumulation in the absence of overt cytotoxicity, therefore allowing to differentiate between cytotoxic and apoptotic effects. Palmitic (C16:0) and oleic (C18:1) acids are the most abundant fatty acids (FFAs) in liver triglycerides in both normal subjects and patients with nonalcoholic fatty liver disease (NAFLD). Therefore, human hepatocytes and HepG2 cells were incubated with a mixture of different proportions of saturated (palmitate) and unsaturated (oleate) FFAs to induce fat-overloading. Similar intracellular levels of lipid accumulation as in the human steatotic liver were achieved. Individual FFAs have a distinct inherent toxic potential. Fat accumulation, cytotoxicity and apoptosis in cells exposed to the FFA mixtures were investigated. The FFA mixture containing a low proportion of palmitic acid (oleate/palmitate, 2:1 ratio) is associated with minor toxic and apoptotic effects, thus representing a cellular model of steatosis that mimics benign chronic steatosis. On the other hand, a high proportion of palmitic acid (oleate/palmitate, 0:3 ratio) might represent a cellular model of steatosis in which saturated FFAs promote an acute harmful effect of fat overaccumulation in the liver. These hepatic cellular models are apparently suitable to experimentally investigate the impact of fat overaccumulation in the liver excluding other factors that could influence hepatocyte behaviour.
Flow cytometry is a single-cell based technology aimed to quantify the scattering of light and the emission of multiple fluorescence signals by individual cells, biological vesicles, or synthetic ...microscopical particles when examined one by one at high speed using lasers or other suitable illumination sources ...
Antibiotic resistance is now a first-order health problem, which makes the development of new families of antimicrobials imperative. These compounds should ideally be inexpensive, readily available, ...highly active, and non-toxic. Here, we present the results of our investigation regarding the antimicrobial activity of a series of natural and synthetic polyamines with different architectures (linear, tripodal, and macrocyclic) and their derivatives with the oxygen-containing aromatic functional groups 1,3-benzodioxol, ortho/para phenol, or 2,3-dihydrobenzofuran. The new compounds were prepared through an inexpensive process, and their activity was tested against selected strains of yeast, as well as Gram-positive and Gram-negative bacteria. In all cases, the conjugated derivatives showed antimicrobial activity higher than the unsubstituted polyamines. Several factors, such as the overall charge at physiological pH, lipophilicity, and the topology of the polyamine scaffold were relevant to their activity. The nature of the lipophilic moiety was also a determinant of human cell toxicity. The lead compounds were found to be bactericidal and fungistatic, and they were synergic with the commercial antifungals fluconazole, cycloheximide, and amphotericin B against the yeast strains tested.
During reproductive life, the human endometrium undergoes around 480 cycles of growth, breakdown and regeneration should pregnancy not be achieved. This outstanding regenerative capacity is the basis ...for women's cycling and its dysfunction may be involved in the etiology of pathological disorders. Therefore, the human endometrial tissue must rely on a remarkable endometrial somatic stem cells (SSC) population. Here we explore the hypothesis that human endometrial side population (SP) cells correspond to somatic stem cells. We isolated, identified and characterized the SP corresponding to the stromal and epithelial compartments using endometrial SP genes signature, immunophenotyping and characteristic telomerase pattern. We analyzed the clonogenic activity of SP cells under hypoxic conditions and the differentiation capacity in vitro to adipogenic and osteogenic lineages. Finally, we demonstrated the functional capability of endometrial SP to develop human endometrium after subcutaneous injection in NOD-SCID mice. Briefly, SP cells of human endometrium from epithelial and stromal compartments display genotypic, phenotypic and functional features of SSC.
Marine mammals may suffer alterations in platelet function and hemostasia due to multiple pathologies, environmental conditions (including stress) or exposure to different contaminants that induce ...platelet activation. Detecting early alterations in platelet function in these animals could be an especially relevant diagnostic tool in these species because they typically do not show signs of weakness or disease until the pathology is in advanced state, in order to avoid attracting predators in natural conditions. The study of early markers of platelet activation is relevant for the detection, monitoring and therapy of inflammation and hemostasis disorders. Flow cytometry provides a convenient method to evaluate platelet activation by following the kinetics of intracellular Ca2+, using sensitive fluorescent indicators that can be loaded into intact cells. In order to study intraplatelet Ca2+ mobilization in marine mammals, we have adapted a kinetic assay of human platelet activation to study platelet activation in whole‐blood samples of bottlenose dolphins (Tursiops truncatus) using the Ca2+‐sensitive dye Fluo‐4AM and a clone of the platelet‐specific antibody CD41‐PE that recognizes dolphin platelets. This no‐wash, no‐lyse protocol provides a simple and sensitive tool to assess in vitro the time course and intensity of signal‐transduction responses to platelet agonists under near‐physiological conditions. The adaptation of this technique to marine mammals represents a methodological advance for basic and clinical veterinary applications but also for general environmental studies on these species.
The detection of reactive oxygen species (ROS) and the analysis of oxidative stress are frequent applications of functional flow cytometry. Identifying and quantifying the ROS species generated ...during oxidative stress are crucial steps for the investigation of molecular mechanisms underlying stress responses. Currently, there is a wide availability of fluorogenic substrates for such purposes, but limitations in their specificity and sensitivity may affect the accuracy of the analysis. The aim of our work was to validate a new experimental model based in different strains of Escherichia coli B deficient in key genes for antioxidant defense, namely oxyR, sodA and sodB. We applied this model to systematically assess issues of specificity in fluorescent probes and the involvement of different ROS in a bacterial model of oxidative stress, as the probes can react with a variety of oxidants and free radical species. Our results confirm the higher sensitivity and specificity of the fluorescent probe mitochondrial peroxy yellow 1 (MitoPY1) for the detection of H2O2, and its very low capacity for organic hydroperoxides, thus extending MitoPY1′s specificity for H2O2 in mammalian cells to a bacterial model. On the contrary, the fluorescent probe 2′,7′-dichlorodihydrofluorescein diacetate (H2DCF-DA) is more sensitive to organic peroxides than to H2O2, confirming the lack of selectivity of H2DCF-DA to H2O2. Treatment with organic peroxides and H2O2 suggests a superoxide-independent oxidation of the fluorescent probe Hydroethidine (HE). We found a positive correlation between the lipophilicity of the peroxides and their toxicity to E. coli, suggesting greater quantitative importance of the peroxidative effects on the bacterial membrane and/or greater efficiency of the protection systems against the intracellular effects of H2O2 than against the membrane oxidative stress induced by organic peroxides. Altogether, our results may aid in preventing or minimizing experimental errors and providing recommendations for the proper design of cytometric studies of oxidative stress, in accordance with current recommendations and guidelines.
A precise understanding of mechanisms used by human embryonic stem cells (hESCs) to maintain genomic integrity is very important for their potential clinical applications. The G1 checkpoint serves to ...protect genomic integrity and prevents cells with damaged DNA from entering S-phase. Previously, we have shown that downregulation of cyclin-dependent kinase 2 (CDK2) in hESC causes G1 arrest, loss of pluripotency, upregulation of cell cycle inhibitors p21 and p27 and differentiation toward extraembryonic lineages. In this study, we investigate in detail the role of CDK2 in cellular processes, which are crucial to the maintenance of genomic stability in hESC such as G1 checkpoint activation, DNA repair, and apoptosis. Our results suggest that downregulation of CDK2 triggers the G1 checkpoint through the activation of the ATM-CHK2-p53-p21 pathway. Downregulation of CDK2 is able to induce sustained DNA damage and to elicit the DNA damage response (DDR) as evidenced by the formation of distinct γ-H2.AX and RAD52-BRCA1 foci in hESC nuclei. CDK2 downregulation causes high apoptosis at the early time points; however, this is gradually decreased overtime as the DDR is initiated. Our mass spectrometry analysis suggest that CDK2 does interact with a large number of proteins that are involved in key cellular processes such as DNA replication, cell cycle progression, DNA repair, chromatin modeling, thus, suggesting a crucial role for CDK2 in orchestrating a fine balance between cellular proliferation, cell death, and DNA repair in hESC.
Oxidative stress is considered a potential etiological factor for Crohn's disease (CD). We characterized the reactive oxygen species (ROS) generated in immune peripheral cells of CD patients, as well ...as their antioxidant enzyme status and the presence of oxidative damage. In addition, mitochondrial function (DeltaPsim) was analyzed to detect the possible origin of ROS.
Cells were obtained from patients at the onset of disease, prior to any treatment. Experiments were repeated when patients were in clinical remission. A set of experiments was carried out in a group of CD patients in persistent morphological remission. Controls were healthy volunteers who were not receiving any treatment at the time. The generation of superoxide, hydrogen peroxide (H(2)O(2)) and nitric oxide, DeltaPsim, superoxide dismutase (SOD) and catalase (CAT) activities, and concentrations of malondyaldehyde (MDA) and 8-oxo-deoxyguanosine (8-oxo-dG) were measured.
SOD activity and H(2)O(2) production were significantly higher during active CD but returned to control levels in remission. DeltaPsim was inhibited during active CD and, although it returned to control levels, its recovery took longer than clinical remission. CAT activity was permanently inhibited during CD, independent of the disease activity. MDA and 8-oxo-dG were permanently elevated.
Oxidative stress during active CD depends on H(2)O(2) production. The inhibition of DeltaPsim suggests that this organelle is a source of ROS. CAT is permanently inhibited in CD, the biological significance of which is under study. The persistent oxidative damage detected may have implications for the evolution of the disease.
Curcumin, a major active component of turmeric (
, L.), is known to have various effects on both healthy and cancerous tissues. In vitro studies suggest that curcumin inhibits cancer cell growth by ...activating apoptosis, but the mechanism underlying the anticancer effect of curcumin is still unclear. Since there is a recent consensus about endoplasmic reticulum (ER) stress being involved in the cytotoxicity of natural compounds, we have investigated using Image flow cytometry the mechanistic aspects of curcumin's destabilization of the ER, but also the status of the lysosomal compartment. Curcumin induces ER stress, thereby causing an unfolded protein response and calcium release, which destabilizes the mitochondrial compartment and induce apoptosis. These events are also associated with secondary lysosomal membrane permeabilization that occurs later together with an activation of caspase-8, mediated by cathepsins and calpains that ended in the disruption of mitochondrial homeostasis. These two pathways of different intensities and momentum converge towards an amplification of cell death. In the present study, curcumin-induced autophagy failed to rescue all cells that underwent type II cell death following initial autophagic processes. However, a small number of cells were rescued (successful autophagy) to give rise to a novel proliferation phase.
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