Semisynthetic Abietic and Dehydroabietic Acid Derivatives and Triptoquinone Epimers Interfere with LPS-Triggered Activation of Dendritic Cells.

Abietic acid (AA), dehydroabietic acid (DHA) and triptoquinones (TQs) are bioactive abietane-type diterpenoids, which are present in many edible vegetables and medicinal herbs with health-promoting properties. Evidence suggests that beneficial effects of diterpenes operate, at least in part, through effects on cells in the immune system. Dendritic cells (DCs) are a key type of leukocyte involved in the initiation and regulation of the immune/inflammatory response and natural or synthetic compounds that modulate DC functions could be potential anti-inflammatory/immunomodulatory agents. Herein, we report the screening of 23 known semisynthetic AA and DHA derivatives, and TQs, synthesized previously by us, in a multi-analyte DC-based assay that detects inhibition of pro-inflammatory cytokine production. Based on the magnitude of the inhibitory effect observed and the number of cytokines inhibited, a variety of activities among compounds were observed, ranging from inactive/weak to very potent inhibitors. Structurally, either alcohol or methyl ester substituents on ring A along with the introduction of aromaticity and oxidation in ring C in the abietane skeleton were found in compounds with higher inhibitory properties. Two DHA derivatives and two TQs exhibited a significant inhibition in all pro-inflammatory cytokines tested and were further investigated. The results confirmed their ability to inhibit, dose dependently, LPS-stimulated expression of the co-stimulatory molecules CD40 and/or CD86 and the production of the pro-inflammatory cytokines IL-1ß, IL-6, IL-12 and TNFα. Our results demonstrate that DC maturation process can be targeted by semisynthetic DHA derivatives and TQ epimers and indicate the potential of these compounds as optimizable anti-inflammatory/immunomodulatory agents.

TGFß lengthens the G1 phase of stem cells in aged mouse brain.

Neurogenesis decreases during aging causing a progressive cognitive decline but it is still controversial whether proliferation defects in neurogenic niches result from a loss of neural stem cells or from an impairment of their progression through the cell cycle. Using an accurate fluorescence-activated cell sorting technique, we show that the pool of neural stem cells is maintained in the subventricular zone of middle-aged mice while they have a reduced proliferative potential eventually leading to the subsequent decrease of their progeny. In addition, we demonstrate that the G1 phase is lengthened during aging specifically in activated stem cells, but not in transit-amplifying cells, and directly impacts on neurogenesis. Finally, we report that inhibition of TGFß signaling restores cell cycle progression defects in stem cells. Our data highlight the significance of cell cycle dysregulation in stem cells in the aged brain and provide an attractive foundation for the development of anti-TGFß regenerative therapies based on stimulating endogenous neural stem cells.

Proatherogenic macrophage activities are targeted by the flavonoid quercetin.

Many studies have demonstrated that the flavonoid quercetin protects against cardiovascular disease (CVD) and related risk factors. Atherosclerosis, the underlying cause of CVD, is also attenuated by oral quercetin administration in animal models. Although macrophages are key players during fatty streak formation and plaque progression and aggravation, little is known about the effects of quercetin on atherogenic macrophages. Here, we report that primary bone marrow-derived macrophages internalized less oxidized low-density lipoprotein (oxLDL) and accumulated less intracellular cholesterol in the presence of quercetin. This reduction of foam cell formation correlated with reduced surface expression of the oxLDL receptor CD36. Quercetin also targeted the lipopolysaccharide-dependent, oxLDL-independent pathway of lipid droplet formation in macrophages. In oxLDL-stimulated macrophages, quercetin inhibited reactive oxygen species production and interleukin (IL)-6 secretion. In a system that evaluated cholesterol crystal-induced IL-1ß secretion via nucleotide-binding domain and leucine-rich repeat containing protein 3 inflammasome activation, quercetin also exhibited an inhibitory effect. Dyslipidemic apolipoprotein E-deficient mice chronically treated with intraperitoneal quercetin injections had smaller atheromatous lesions, reduced lipid deposition, and less macrophage and T cell inflammatory infiltrate in the aortic roots than vehicle-treated animals. Serum levels of total cholesterol and the lipid peroxidation product malondialdehyde were also reduced in these mice. Our results demonstrate that quercetin interferes with both key proatherogenic activities of macrophages, namely foam cell formation and pro-oxidant/proinflammatory responses, and these effects may explain the atheroprotective properties of this common flavonoid.

Alternative lengthening of telomeres in human glioma stem cells.

Cancer stem cells are increasingly recognized as major therapeutic targets. We report here the isolation of glioma stem cells (GSCs) maintaining telomere length through a telomerase-independent mechanism known as alternative lengthening of telomeres (ALTs). TG20 cells were isolated from a glioblastoma multiforme, which had the ALT phenotype. They have no detectable telomerase activity and extremely long and heterogeneous telomeres colocalizing with promyelocytic leukemia bodies. The cancer stem cell potential of TG20 cells was confirmed based on their expression of neural stem cell markers, their capacity of in vitro long-term proliferation and to form intracranial tumors in immune-deficient mice. Interestingly, we found that both in vitro and in vivo TG20 cells were significantly more resistant to ionizing radiation than GSCs with telomerase activity. Analysis of DNA damage foci, DNA double-strand breaks repair, and chromosome instability suggest that radiation resistance was related to interference of ALT pathway with DNA damage response. Therefore, our data show for the first time that the ALT pathway can confer to cancer stem cells the capacity to sustain long-term proliferation as telomerase activity and importantly may also affect treatment efficiency. TG20 cells are thus the first cellular model of GSCs displaying ALT and should prove to be useful for the development of specific treatment strategies.

A Mouse Model of Ulcerative Cutaneous Leishmaniasis by Leishmania (Viannia) panamensis to Investigate Infection, Pathogenesis, Immunity, and Therapeutics.

A mouse model of cutaneous leishmaniasis (CL) by Leishmania (Viannia) panamensis (L(V)p) that reproduces the characteristics of the human disease remains elusive. Here we report the development of a CL model that uses a mouse-adapted L(V)p isolate to reproducibly induce a dermal disease with a remarkable similarity to human CL. BALB/c mice infected intradermally in the ear with 105 stationary UA-946 L(V)p promastigotes develop a progressive cutaneous disease that exhibits the typical ulcerated lesions with indurated borders observed in CL patients. Although most of parasites in the inoculum die within the first week of infection, the survivors vigorously multiply at the infection site during the following weeks, paralleling disease appearance and aggravation. Regional lymphadenopathy as well as lymphatic dissemination of parasites to draining lymph nodes (dLN) was evidenced early after infection. Viable parasites were also isolated from spleen at later timepoints indicating systemic parasitic dissemination, but, strikingly, no signs of systemic disease were observed. Increasing numbers of myeloid cells and T lymphocytes producing IFNγ and IL-4 were observed in the dLN as disease progressed. A mixed adaptive L(V)p-specific T cell-mediated response was induced, since ex vivo recall experiments using dLN cells and splenocytes revealed the production of type 1 (IFNγ, IL-2), type 2 (IL-4, IL-13), regulatory (IL-10), and inflammatory (GM-CSF, IL-3) cytokines. Humoral adaptive response was characterized by early production of IgG1- followed by IgG2a-type of L(V)p-specific antibodies. IFNγ/IL-4 and IgG2a/IgG1 ratios indicated that the initial non-protective Th2 response was redirected toward a protective Th1 response. In situ studies revealed a profuse recruitment of myeloid cells and of IFNγ- and IL-4-producing T lymphocytes to the site of infection, and the typical histopathological changes induced by dermotropic Leishmania species. Evidence that this model is suitable to investigate pharmacological and immunomodulatory interventions, as well as for antigen discovery and vaccine development, is also presented. Altogether, these results support the validity and utility of this novel mouse model to study the pathogenesis, immunity, and therapeutics of L(V)p infections.

Stem and Cancer Stem Cell Identities, Cellular Markers, Niche Environment and Response to Treatments to Unravel New Therapeutic Targets.

Adult stem cells are a partially quiescent cell population responsible for natural cell renewal and are found in many different regions of the body, including the brain, teeth, bones, muscles, skin, and diverse epithelia, such as the epidermal or intestinal epithelium, among others [...].

IL-10-Dependent Amelioration of Chronic Inflammatory Disease by Microdose Subcutaneous Delivery of a Prototypic Immunoregulatory Small Molecule.

One of the interventional strategies to reestablish the immune effector/regulatory balance, that is typically altered in chronic inflammatory diseases (CID), is the reinforcement of endogenous immunomodulatory pathways as the one triggered by interleukin (IL)-10. In a recent work, we demonstrated that the subcutaneous (sc) administration of an IL-10/Treg-inducing small molecule-based formulation, using a repetitive microdose (REMID) treatment strategy to preferentially direct the effects to the regional immune system, delays the progression of atherosclerosis. Here we investigated whether the same approach using other IL-10-inducing small molecule, such as the safe, inexpensive, and widely available polyphenol curcumin, could induce a similar protective effect in two different CID models. We found that, in apolipoprotein E deficient mice, sc treatment with curcumin following the REMID strategy induced atheroprotection that was not consequence of its direct systemic lipid-modifying or antioxidant activity, but instead paralleled immunomodulatory effects, such as reduced proatherogenic IFNγ/TNFα-producing cells and increased atheroprotective FOXP3+ Tregs and IL-10-producing dendritic and B cells. Remarkably, when a similar strategy was used in the neuroinflammatory model of experimental autoimmune encephalomyelitis (EAE), significant clinical and histopathological protective effects were evidenced, and these were related to an improved effector/regulatory cytokine balance in restimulated splenocytes. The essential role of curcumin-induced IL-10 for neuroprotection was confirmed by the complete abrogation of the clinical effects in IL-10-deficient mice. Finally, the translational therapeutic prospection of this strategy was evidenced by the neuroprotection observed in mice starting the treatment one week after disease triggering. Collectively, results demonstrate the power of a simple natural IL-10-inducing small molecule to tackle chronic inflammation, when its classical systemic and direct pharmacological view is shifted towards the targeting of regional immune cells, in order to rationally harness its immunopharmacological potential. This shift implies that many well-known IL-10-inducing small molecules could be easily reformulated and repurposed to develop safe, innovative, and accessible immune-based interventions for CID.

Vasculogenesis from Human Dental Pulp Stem Cells Grown in Matrigel with Fully Defined Serum-Free Culture Media.

The generation of vasculature is one of the most important challenges in tissue engineering and regeneration. Human dental pulp stem cells (hDPSCs) are some of the most promising stem cell types to induce vasculogenesis and angiogenesis as they not only secrete vascular endothelial growth factor (VEGF) but can also differentiate in vitro into both endotheliocytes and pericytes in serum-free culture media. Moreover, hDPSCs can generate complete blood vessels containing both endothelial and mural layers in vivo, upon transplantation into the adult brain. However, many of the serum free media employed for the growth of hDPSCs contain supplements of an undisclosed composition. This generates uncertainty as to which of its precise components are necessary and which are dispensable for the vascular differentiation of hDPSCs, and also hinders the transfer of basic research findings to clinical cell therapy. In this work, we designed and tested new endothelial differentiation media with a fully defined composition using standard basal culture media supplemented with a mixture of B27, heparin and growth factors, including VEGF-A165 at different concentrations. We also optimized an in vitro Matrigel assay to characterize both the ability of hDPSCs to differentiate to vascular cells and their capacity to generate vascular tubules in 3D cultures. The description of a fully defined serum-free culture medium for the induction of vasculogenesis using human adult stem cells highlights its potential as a relevant innovation for tissue engineering applications. In conclusion, we achieved efficient vasculogenesis starting from hDPSCs using serum-free culture media with a fully defined composition, which is applicable for human cell therapy purposes.

Reformulating Small Molecules for Cardiovascular Disease Immune Intervention: Low-Dose Combined Vitamin D/Dexamethasone Promotes IL-10 Production and Atheroprotection in Dyslipidemic Mice.

The targeting of proinflammatory pathways has a prophylactic and therapeutic potential on atherosclerotic cardiovascular diseases (CVD). An alternative/complementary strategy is the promotion of endogenous atheroprotective mechanisms that are impaired during atherosclerosis progression, such as the activity of tolerogenic dendritic cells (tolDC) and regulatory T cells (Treg). There is a need to develop novel low cost, safe and effective tolDC/Treg-inducing formulations that are atheroprotective and that can be of easy translation into clinical settings. We found that apolipoprotein E-deficient (ApoE-/-) mice treated with a low-dose combined formulation of Vitamin D and Dexamethasone (VitD/Dexa), delivered repetitively and subcutaneously (sc) promoted interleukin-10 (IL-10) production by dendritic cells and other antigen presenting cells in the lymph nodes draining the site of injection and the spleens. Expectedly, the treatment also increased the numbers of IL-10-producing CD4+ T cells. Concomitantly, the frequency of IFNγ-producing CD4+ and CD8+ T cells in the spleen, and the IFNγ response of splenocytes to polyclonal stimulation ex vivo were lower after VitD/Dexa treatment, indicating a reduced proatherogenic Th1 response. Interestingly, VitD/Dexa-treated mice had smaller atherosclerotic lesions, with reduced lipid content and lower inflammatory infiltrate of macrophages and T cells in the aortic root. No hypolipidemic or antioxidant effect could be detected, suggesting that a dominantly immunomodulatory mechanism of atheroprotection was engaged under the low-dose sc VitD/Dexa conditions used. Finally, no evidence of clinical, biochemical or immune toxicity was observed in treated ApoE-/- mice and, most importantly, C57BL/6 mice latently infected with Leishmania parasites and treated with an identical VitD/Dexa dose/scheme showed no clinical or microbiological signs of disease reactivation, suggesting the absence of general immunosuppression. Altogether, these results indicate that a non-toxic, non-immunosuppressive, low-dose of VitD/Dexa, administered subcutaneously and repetitively, exerts atheroprotective effects in dyslipidemic mice, apparently due to the induction of an IL-10-producing network of lymphoid and myeloid immune cells. These well known, widely available, and inexpensive small molecules can be easily co-formulated into a simple and accessible agent with a potential use as a prophylactic or therapeutic immune intervention for CVD and other chronic inflammatory diseases.

The HIF1α/JMY pathway promotes glioblastoma stem-like cell invasiveness after irradiation.

Human glioblastoma (GBM) is the most common primary malignant brain tumor. A minor subpopulation of cancer cells, known as glioma stem-like cells (GSCs), are thought to play a major role in tumor relapse due to their stem cell-like properties, their high resistance to conventional treatments and their high invasion capacity. We show that ionizing radiation specifically enhances the motility and invasiveness of human GSCs through the stabilization and nuclear accumulation of the hypoxia-inducible factor 1α (HIF1α), which in turn transcriptionally activates the Junction-mediating and regulatory protein (JMY). Finally, JMY accumulates in the cytoplasm where it stimulates GSC migration via its actin nucleation-promoting activity. Targeting JMY could thus open the way to the development of new therapeutic strategies to improve the efficacy of radiotherapy and prevent glioma recurrence.

Cystamine and cysteamine increase brain levels of BDNF in Huntington disease via HSJ1b and transglutaminase.

There is no treatment for the neurodegenerative disorder Huntington disease (HD). Cystamine is a candidate drug; however, the mechanisms by which it operates remain unclear. We show here that cystamine increases levels of the heat shock DnaJ-containing protein 1b (HSJ1b) that are low in HD patients. HSJ1b inhibits polyQ-huntingtin-induced death of striatal neurons and neuronal dysfunction in Caenorhabditis elegans. This neuroprotective effect involves stimulation of the secretory pathway through formation of clathrin-coated vesicles containing brain-derived neurotrophic factor (BDNF). Cystamine increases BDNF secretion from the Golgi region that is blocked by reducing HSJ1b levels or by overexpressing transglutaminase. We demonstrate that cysteamine, the FDA-approved reduced form of cystamine, is neuroprotective in HD mice by increasing BDNF levels in brain. Finally, cysteamine increases serum levels of BDNF in mouse and primate models of HD. Therefore, cysteamine is a potential treatment for HD, and serum BDNF levels can be used as a biomarker for drug efficacy.

Rapid differentiation of isobaric and positional isomers of structurally related glycosides from Phytolacca bogotensis.

Through the action of glycosyltransferases, a plant can biosynthetically assemble small different aglycons or 'templates' to various polysaccharides to produce numerous glycoconjugates differing in the type of the attached aglycon, the anomeric configuration of C-1 of the glycosylating sugar, the type of sugar and the different position of attachments of the sugar unit present in the polysaccharide chain. The position of attachments and the anomeric configuration of the different sugar present in the polysaccharide create the opportunity to generate molecules with either the same or very close molecular weights, which have relative structural similarity--forming isobaric and positional isomers. Although isomeric differentiation was once considered outside of the domain of mass spectrometry, this task can now be resolved using tandem mass spectrometry. In a standardized purified glycoconjugate fraction (SPT01) from Phytolacca bogotensis, we report conventional electrospray ionization mass spectrometry and collision-induced dissociation (CID) MS/MS parameters which favored the formation of characteristic product ions. This allowed us to suggest the type of sugar linkages present in a specific glycoconjugate. Ten new glycoconjugate are described from this plant and another twelve known saponins were structurally characterized using the automatic MSn acquisition mode. The differentiation of two pairs of positional isomers and four isobaric glycosides and the production of a library of 30 glycosides present in P. bogotensis were accomplished.

Green Coffee Extract Improves Cardiometabolic Parameters and Modulates Gut Microbiota in High-Fat-Diet-Fed ApoE-/- Mice.

Chlorogenic acids (CGA) are the most abundant phenolic compounds in green coffee beans and in the human diet and have been suggested to mitigate several cardiometabolic risk factors. Here, we aimed to evaluate the effect of a water-based standardized green coffee extract (GCE) on cardiometabolic parameters in ApoE-/- mice and to explore the potential underlying mechanisms. Mice were fed an atherogenic diet without (vehicle) or with GCE by gavage (equivalent to 220 mg/kg of CGA) for 14 weeks. We assessed several metabolic, pathological, and inflammatory parameters and inferred gut microbiota composition, diversity, and functional potential. Although GCE did not reduce atherosclerotic lesion progression or plasma lipid levels, it induced important favorable changes. Specifically, improved metabolic parameters, including fasting glucose, insulin resistance, serum leptin, urinary catecholamines, and liver triglycerides, were observed. These changes were accompanied by reduced weight gain, decreased adiposity, lower inflammatory infiltrate in adipose tissue, and protection against liver damage. Interestingly, GCE also modulated hepatic IL-6 and total serum IgM and induced shifts in gut microbiota. Altogether, our results reveal the cooccurrence of these beneficial cardiometabolic effects in response to GCE in the same experimental model and suggest potential mediators and pathways involved.

Genomic Analysis of Colombian Leishmania panamensis strains with different level of virulence.

The establishment of Leishmania infection in mammalian hosts and the subsequent manifestation of clinical symptoms require internalization into macrophages, immune evasion and parasite survival and replication. Although many of the genes involved in these processes have been described, the genetic and genomic variability associated to differences in virulence is largely unknown. Here we present the genomic variation of four Leishmania (Viannia) panamensis strains exhibiting different levels of virulence in BALB/c mice and its application to predict novel genes related to virulence. De novo DNA sequencing and assembly of the most virulent strain allowed comparative genomics analysis with sequenced L. (Viannia) panamensis and L. (Viannia) braziliensis strains, and showed important variations at intra and interspecific levels. Moreover, the mutation detection and a CNV search revealed both base and structural genomic variation within the species. Interestingly, we found differences in the copy number and protein diversity of some genes previously related to virulence. Several machine-learning approaches were applied to combine previous knowledge with features derived from genomic variation and predict a curated set of 66 novel genes related to virulence. These genes can be prioritized for validation experiments and could potentially become promising drug and immune targets for the development of novel prophylactic and therapeutic interventions.

Opposite effects of GCN5 and PCAF knockdowns on the alternative mechanism of telomere maintenance.

Cancer cells can use a telomerase-independent mechanism, known as alternative lengthening of telomeres (ALT), to elongate their telomeres. General control non-derepressible 5 (GCN5) and P300/CBP-associated factor (PCAF) are two homologous acetyltransferases that are mutually exclusive subunits in SAGA-like complexes. Here, we reveal that down regulation of GCN5 and PCAF had differential effects on some phenotypic characteristics of ALT cells. Our results suggest that GCN5 is present at telomeres and opposes telomere recombination, in contrast to PCAF that may indirectly favour them in ALT cells.

Interleukin-10 production and T cell-suppressive capacity in B cell subsets from atherosclerotic apoE -/- mice.

The evidence regarding the role of regulatory B cells (Breg) in atherosclerosis are scarce, and there are contradictory data about their atheroprotective properties. Due to the demonstrated protective function of Breg in different inflammatory diseases mainly through interleukin-10 (IL-10) production, the knowledge of their participation in atherosclerosis immunopathology would be very valuable. To further study which B cell subsets participate in IL-10 production and their regulatory role, splenocytes from apolipoprotein-E-deficient mice were evaluated by ex vivo and in vitro cultures. Atherosclerotic mice had increased frequency of IL-10+ B cells, which presented high CD1d, CD19, and IgM, but variable CD5, CD21, and CD23 expression. IL-10+ B cells were not enriched in B cell subsets previously reported as Breg. Increased frequency of IL-10+ B cells with transitional 1-like (T1-like) and follicular (FO) and reduced CD5+ and marginal zone (MZ) phenotypes were observed ex vivo. Increased frequency of IL-10+ B cells with T1-like and MZ, and decreased IL-10+ FO and T2 phenotypes were also observed in vitro. To determine regulatory capacity of B cells in the atherosclerotic model, each subset were co-cultured with CD4+CD25- T cells. CD5+, FO, MZ, and T1-like cells from atherosclerotic mice exhibited regulation in an IL-10-dependent manner. However, only FO cells decreased both frequency of interferon gamma (IFN-γ)+ and tumor necrosis factor alpha (TNF-α)+ and proliferation of T cells. Finally, splenocytes showed increased frequency of IFN-γ+ and TNF-α+ cells only when FO-depleted B cells were evaluated. These results suggest that mainly FO B cells can modulate in some level the inflammatory responses observed in atherosclerosis.

Natural Biflavonoids Modulate Macrophage-Oxidized LDL Interaction In Vitro and Promote Atheroprotection In Vivo.

The accumulation of oxidized ApoB-100-containing lipoproteins in the vascular intima and its subsequent recognition by macrophages results in foam cell formation and inflammation, key events during atherosclerosis development. Agents targeting this process are considered potentially atheroprotective. Since natural biflavonoids exert antioxidant and anti-inflammatory effects, we evaluated the atheroprotective effect of biflavonoids obtained from the tropical fruit tree Garcinia madruno. To this end, the pure biflavonoid aglycones morelloflavone (Mo) and volkensiflavone (Vo), as well as the morelloflavone's glycoside fukugiside (Fu) were tested in vitro in primary macrophages, whereas a biflavonoid fraction with defined composition (85% Mo, 10% Vo, and 5% Amentoflavone) was tested in vitro and in vivo. All biflavonoid preparations were potent reactive oxygen species (ROS) scavengers in the oxygen radical absorbance capacity assay, and most importantly, protected low-density lipoprotein particle from both lipid and protein oxidation. In biflavonoid-treated macrophages, the surface expression of the oxidized LDL (oxLDL) receptor CD36 was significantly lower than in vehicle-treated macrophages. Uptake of fluorescently labeled oxLDL and cholesterol accumulation were also attenuated in biflavonoid-treated macrophages and followed a pattern that paralleled that of CD36 surface expression. Fu and Vo inhibited oxLDL-induced ROS production and interleukin (IL)-6 secretion, respectively, whereas all aglycones, but not the glucoside Fu, inhibited the secretion of one or more of the cytokines IL-1ß, IL-12p70, and monocyte chemotactic protein-1 (MCP-1) in lipopolysaccharide (LPS)-stimulated macrophages. Interestingly, in macrophages primed with low-dose LPS and stimulated with cholesterol crystals, IL-1ß secretion was significantly and comparably inhibited by all biflavonoid preparations. Intraperitoneal administration of the defined biflavonoid fraction into ApoE-/- mice was atheroprotective, as evidenced by the reduction of the atheromatous lesion size and the density of T cells and macrophages infiltrating the aortic root; moreover, this treatment also lowered the circulating levels of cholesterol and the lipid peroxidation product malondialdehyde. These results reveal the potent atheroprotective effects exerted by biflavonoids on key events of the oxLDL-macrophage interphase: (i) atheroligand formation, (ii) atheroreceptor expression, (iii) foam cell transformation, and (iv) prooxidant/proinflammatory macrophage response. Furthermore, our results also evidence the antioxidant, anti-inflammatory, hypolipemiant, and atheroprotective effects of Garcinia madruno's biflavonoids in vivo.