Cocktail Of Mediators Research Articles

764 Targeting dermal fibroblast senescence with senomorphic and epigenetic properties of haritaki fruit extract: Effects on SASP and miR over-expression in senescent fibroblasts’ extracellular vesicles

Skin aging is the consequence of two biological process: intrinsic genetically programmed factors and extrinsic environmental factors. At the cellular level, accumulation of senescent cells (SC) is also involved in skin aging. SC stop to proliferate but remain metabolically active. The secretome of SC (Senescence Associated Secretory Phenotype: SASP), is a cocktail of mediators like CSF3, CXCL1, IL8, IL1b, IL6, MMP-1 and -3, involved in skin aging. Extracellular vesicles (EV) have also been reported to act as cytokines during intercellular communication. They transport proteins, mRNAs and miRNAs, over short or long distances. miRNAs are a class of short, single-stranded, noncoding RNA molecules. As epigenetic modulators, miRNAs affect the protein levels of the target mRNAs without modifying the gene sequences. Several miRs are known to be involved in senescence and skin aging. The aim of our study was to evaluate, the effect of Haritaki fruit extract (HF extract) on SASP and senescence associated miRs contained in EV. Our results showed that, 14 days after ionizing radiation (7.5 Gy), human dermal fibroblasts presented a senescent phenotype: flattened and irregular shape, senescence associated ß-galactosidase activity, SASP expression (RT-PCR) and miR 24a-3p, miR 186-5p, miR 30a-3p and miR 29a-3p overexpression in EV (RT-PCR). Our results also showed that incubation of senescent fibroblasts with HF extract strongly decreased SASP mRNA level and miR 34a-5p, miR 24a-3p, miR 186-5p, miR 30a-3p and miR 29a-3p over-expression in EV. Taken together these results demonstrated that Haritaki fruit extract is a strong senomorphic agent with epigenetic effect, suggesting that it may be useful for the development of new anti-aging dermo-cosmetic products.

Laccase Mediator Cocktail System as a Sustainable Skin Whitening Agent for Deep Eumelanin Decolorization.

The overproduction of eumelanin leads to a panel of unaesthetic hyper-pigmented skin diseases, including melasma and age spots. The treatment of these diseases often requires the use of tyrosinase inhibitors, which act as skin whitening agents by inhibiting the synthesis of eumelanin, with harmful side effects. We report here that laccase from Trametes versicolor in association with a cocktail of natural phenol redox mediators efficiently degraded eumelanin from Sepia officinalis, offering an alternative procedure to traditional whitening agents. Redox mediators showed a synergistic effect with respect to their single-mediator counterpart, highlighting the beneficial role of the cocktail system. The pro-oxidant DHICA sub-units of eumelanin were degraded better than the DHI counterpart, as monitored by the formation of pyrrole-2,3,5-tricarboxylic acid (PTCA) and pyrrole-2,3-dicarboxylic acid (PDCA) degradation products. The most effective laccase-mediated cocktail system was successively applied in a two-component prototype of a topical whitening cream, showing high degradative efficacy against eumelanin.

Immune system and atherosclerosis: Hostile or friendly relationship.

Coronary artery disease has remained a major health challenge despite enormous progress in prevention, diagnosis, and treatment strategies. Formation of atherosclerotic plaque is a chronic process that is developmentally influenced by intrinsic and extrinsic determinants. Inflammation triggers atherosclerosis, and the fundamental element of inflammation is the immune system. The immune system involves in the atherosclerosis process by a variety of immune cells and a cocktail of mediators. It is believed that almost all main components of this system possess a profound contribution to the atherosclerosis. However, they play contradictory roles, either protective or progressive, in different stages of atherosclerosis progression. It is evident that monocytes are the first immune cells appeared in the atherosclerotic lesion. With the plaque growth, other types of the immune cells such as mast cells, and T lymphocytes are gradually involved. Each cell releases several cytokines which cause the recruitment of other immune cells to the lesion site. This is followed by affecting the expression of other cytokines as well as altering certain signaling pathways. All in all, a mix of intertwined interactions determine the final outcome in terms of mild or severe manifestations, either clinical or subclinical. Therefore, it is of utmost importance to precisely understand the kind and degree of contribution which is made by each immune component in order to stop the growing burden of cardiovascular morbidity and mortality. In this review, we present a comprehensive appraisal on the role of immune cells in the atherosclerosis initiation and development.

373 Effect of Haritaki fruit extract on Senescence Associated Secretory Phenotype and miR 30a-3p over-expression in senescent dermal fibroblasts’ extracellular vesicles

Skin aging is the consequence of two biological process: intrinsic genetically programmed factors and extrinsic environmental factors (sun exposure or lifestyle). At the cellular level, accumulation of senescent cells (SC) is also involved in skin aging. SC stop to proliferate but remain metabolically active. The secretome of SC (Senescence Associated Secretory Phenotype: SASP), is a cocktail of mediators (CSF3, CXCL1, IL8, IL1b, IL6, MMP-1 and -3) involved in skin aging. Extracellular vesicles (EV) have also been reported to act in a similar manner as cytokines during intercellular communication. They transport proteins, mRNAs and miRNAs, over short or long distances. miRNAs are a class of short, single-stranded, noncoding RNA molecules. By binding the specific 3’-UTRs sequence of the target mRNAs, they regulate the expression of multiple genes at the post-transcriptional level through degradation. For instance, miR 30a has been described as a key regulator of aging in human epidermis by regulating barrier function and keratinocytes apoptosis in human epidermis. The aim of our study was to evaluate, by RT-PCR, the effect of Haritaki fruit extract (HF extract) on SASP and senescence associated miR 30a-3p contained in EV. Our results showed that, 14 days after ionizing radiation (7.5 Gy), human dermal fibroblasts presented a senescent phenotype: flattened and irregular shape, senescence associated b-galactosidase activity, SASP over-expression (RT-PCR) and mir 30a-3p overexpression in EV (RT-PCR). Our results also showed that incubation of senescent fibroblasts with HF extract strongly decreased SASP mRNA level and miR 30a-3p expression in EV. Taken together these results demonstrated that Haritaki fruit extract is a strong senomorphic agent, suggesting that it may be useful for the development of new anti- aging dermo-cosmetic products.

Mast Cell Changes the Phenotype of Microglia via Histamine and ATP

Microglia are the dynamic motile phagocytes of the brain considered the first line of defense against threats or disturbances to the Central Nervous System (CNS). Microglia help orchestrate the immunological response by interacting with others immune cells. Mast cells (MCs) are effector cells of the innate immune system distributed in all organs and vascularized tissues, brain included. Several molecular mechanisms for potential interactions between MCs and microglia have been determined. However, the effect of MCs on regulated exocytosis and phagocytic clearance in microglia has not been explored. Cocktails of MCs mediators (MCM) obtained at 37°C and 53°C were used to induce microglia activation. Changes in intracellular calcium [Ca2+]i and ATP release were studied by calcium and quinacrine fluorescence imaging. Fluorescent latex beads were used to assay phagocytosis in microglia after MCM treatment and compared to that measured in the presence of histamine, ATP and lipopolysaccharide (LPS). Iba-1 expression and area were quantified by immunofluorescence and histamine levels evaluated by ELISA techniques. Local application onto microglia of the MC mediator cocktail elicited Ca2+ transients and exocytotic release associated with quinacrine dye de-staining. Ca2+ signals were mimicked by histamine and blocked by the H1 receptor (H1R) antagonist, cetirizine. Hydrolysis of ATP by apyrase also affected Ca2+ transients to a lesser extent. Iba-1 fluorescence, cell area and phagocytosis were enhanced by histamine through H1R. However, ATP prevented iba-1 expression and microglial phagocytosis. MCM showed combined effects of histamine and ATP, increasing the number of internalized microbeads per cell and area without raising iba1 expression. Our results highlight the relevance of MC-derived histamine and ATP in the modulation of secretory and phagocytic activities that would explain the heterogeneity of microglial responses in different pathological contexts.

Proteoglycans involved in bidirectional communication between mast cells and hippocampal neurons

BackgroundMast cells (MCs) in the brain can respond to environmental cues and relay signals to neurons that may directly influence neuronal electrical activity, calcium signaling, and neurotransmission. MCs also express receptors for neurotransmitters and consequently can be activated by them. Here, we developed a coculture model of peritoneal MCs, incubated together with dissociated hippocampal neurons for the study of cellular mechanisms involved in the mast cell-neuron interactions.MethodsCalcium imaging was used to simultaneously record changes in intracellular calcium [Ca2+]i in neurons and MCs. To provide insight into the contribution of MCs on neurotransmitter release in rat hippocampal neurons, we used analysis of FM dye release, evoked by a cocktail of mediators from MCs stimulated by heat.ResultsBidirectional communication is set up between MCs and hippocampal neurons. Neuronal depolarization caused intracellular calcium [Ca2+]i oscillations in MCs that produced a quick response in neurons. Furthermore, activation of MCs with antigen or the secretagogue compound 48/80 also resulted in a neuronal [Ca2+]i response. Moreover, local application onto neurons of the MC mediator cocktail elicited Ca2+ transients and a synaptic release associated with FM dye destaining. Neuronal response was partially blocked by D-APV, a N-methyl-D-aspartate receptor (NMDAR) antagonist, and was inhibited when the cocktail was pre-digested with chondroitinase ABC, which induces enzymatic removal of proteoglycans of chondroitin sulfate (CS).ConclusionsMC-hippocampal neuron interaction affects neuronal [Ca2+]i and exocytosis signaling through a NMDAR-dependent mechanism.

Regulation of FOXP3 expression in myeloid cells in response to all-trans-retinoic acid, interleukin 2 and transforming growth factor β

FoxP3 is a transcription factor essential for differentiation and function of T regulatory cells (Tregs). There are two major subsets of Tregs: natural Tregs (nTregs) generated in thymus and inducible Tregs (iTregs) produced in peripheral immune system. It has been documented that iTreg development is dependent on soluble mediators including interleukin 2 (IL2), transforming growth factor β (TGFβ) and all-trans-retinoic acid (ATRA). In our experiments we performed a gene expression array, followed by Real-time PCR experiments to study expression of genes regulated by ATRA in cells of myeloid origin. Our experiments revealed that ATRA alone, but also a cocktail of mediators consisting of IL2, TGFβ and ATRA, upregulate expression of FOXP3 gene in normal and leukemic myeloid cells. Our results indicate that signaling pathways which are used at the late steps of T cell differentiation, are also active in the cells of myeloid lineage.

The development of CotA mediator cocktail system for dyes decolorization.

The increasing use of dyes leads to serious environmental concerns, it is significant to explore eco-friendly and economic approaches for dye decolorization. This study aimed to develop mediator cocktail (AS and ABTS) for enhancing the capability of laccase-mediator system in the removal of dyes. By mediator screening, the mediators of ABTS and AS (ABTS, 2, 2'-azino-bis-(3-ethylbenzothiazo-thiazoline-6-sulphonic acid); AS, acetosyringone) were combined for dyes decolorization. The Box-Behnken Design and response surface analysis was performed to optimize experiment conditions. Comparing the CotA-ABTS-AS cocktail system with CotA-single mediator system showed that the coupling of ABTS and AS could increase the decolorization rate 15 times higher, save a third of the cost and shorten the reaction time by 50%. In addition, our studies revealed that sequential oxidation may occur in CotA-ABTS-AS system. Compared with CotA laccase-single mediator system, the CotA-ABTS-AS cocktail system showed advantages including higher efficiency, lower cost and shorter reaction time. This was the first report on the dyes decolorization by laccase mediator cocktail system. These results paved the curb for the application of laccase mediator system in various industrial processes.

Reduced Responses of Submucous Neurons from Irritable Bowel Syndrome Patients to a Cocktail Containing Histamine, Serotonin, TNFα, and Tryptase (IBS-Cocktail).

Background and Aims: Malfunctions of enteric neurons are believed to play an important role in the pathophysiology of irritable bowel syndrome (IBS). Our aim was to investigate whether neuronal activity in biopsies from IBS patients is altered in comparison to healthy controls (HC).Methods: Activity of human submucous neurons in response to electrical nerve stimulation and local application of nicotine or a mixture of histamine, serotonin, tryptase, and TNF-α (IBS-cocktail) was recorded in biopsies from 17 HC and 35 IBS patients with the calcium-sensitive-dye Fluo-4 AM. The concentrations of the mediators resembeled those found in biopsy supernatants or blood. Neuronal activity in guinea-pig submucous neurons was studied with the voltage-sensitive-dye di-8-ANEPPS.Results: Activity in submucous ganglia in response to nicotine or electrical nerve stimulation was not different between HC and IBS patients (P = 0.097 or P = 0.448). However, the neuronal response after application of the IBS-cocktail was significantly decreased (P = 0.039) independent of whether diarrhea (n = 12), constipation (n = 5) or bloating (n = 5) was the predominant symptom. In agreement with this we found that responses of submucous ganglia conditioned by overnight incubation with IBS mucosal biopsy supernatant to spritz application of this supernatant was significantly reduced (P = 0.019) when compared to incubation with HC supernatant.Conclusion: We demonstrated for the first time reduced neuronal responses in mucosal IBS biopsies to an IBS mediator cocktail. While excitability to classical stimuli of enteric neurons was comparable to HC, the activation by the IBS-cocktail was decreased. This was very likely due to desensitization to mediators constantly released by mucosal and immune cells in the gut wall of IBS patients.

Prostate cancer and neuroendocrine differentiation: more neuronal, less endocrine?

Neuroendocrine differentiation (NED) marks a structural and functional feature of certain cancers, including prostate cancer (PCa), whereby the malignant tissue contains a significant proportion of cells displaying neuronal, endocrine, or mixed features. NED cells produce, and can secrete, a cocktail of mediators commonly encountered in the nervous system, which may stimulate and coordinate cancer growth. In PCa, NED appears during advanced stages, subsequent to treatment, and accompanies treatment resistance and poor prognosis. However, the term “neuroendocrine” in this context is intrinsically vague. This article seeks to provide a framework on which a unified view of NED might emerge. First, we review the mutually beneficial interplay between PCa and neural structures, mainly supported by cell biology experiments and neurological conditions. Next, we address the correlations between PCa and neural functions, as described in the literature. Based upon the integration of clinical and basic observations, we suggest that it is legitimate to seek for true neural differentiation, or neuromimicry, in cancer progression, most notably in PCa cells exhibiting what is commonly described as NED.

Listeria monocytogenes Alters Mast Cell Phenotype, Mediator and Osteopontin Secretion in a Listeriolysin-Dependent Manner

Whilst mast cells participate in the immune defence against the intracellular bacterium Listeria monocytogenes, there is conflicting evidence regarding the ability of L. monocytogenes to infect mast cells. It is known that the pore-forming toxin listeriolysin (LLO) is important for mast cell activation, degranulation and the release of pro-inflammatory cytokines. Mast cells, however, are a potential source of a wide range of cytokines, chemokines and other mediators including osteopontin, which contributes to the clearing of L. monocytogenes infections in vivo, although its source is unknown. We therefore aimed to resolve the controversy of mast cell infection by L. monocytogenes and investigated the extent of mediator release in response to the bacterium. In this paper we show that the infection of bone marrow-derived mast cells by L. monocytogenes is inefficient and LLO-independent. LLO, however, is required for calcium-independent mast cell degranulation as well as for the transient and selective downregulation of cell surface CD117 (c-kit) on mast cells. We demonstrate that in addition to the key pro-inflammatory cytokines TNF-α and IL-6, mast cells release a wide range of other mediators in response to L. monocytogenes. Osteopontin, IL-2, IL-4, IL-13 and granulocyte macrophage colony-stimulating factor (GM-CSF), and chemokines including CCL2, CCL3, CCL4 and CCL5 are released in a MyD88-dependent manner. The wide range of mediators released by mast cells in response to L. monocytogenes may play an important role in the recruitment and activation of a variety of immune cells in vivo. The cocktail of mediators, however, is unlikely to skew the immune response to a particular effector response. We propose that mast cells provide a hitherto unreported source of osteopontin, and may provide an important role in co-ordinating the immune response during Listeria infection.

Functions and Imaging of Mast Cell and Neural Axis of the Gut

Close association between nerves and mast cells in the gut wall provides the microanatomic basis for functional interactions between these elements, supporting the hypothesis that a mast cell-nerve axis influences gut functions in health and disease. Advanced morphology and imaging techniques are now available to assess structural and functional relationships of the mast cell-nerve axis in human gut tissues. Morphologic techniques including co-labeling of mast cells and nerves serve to evaluate changes in their densities and anatomic proximity. Calcium (Ca(++)) and potentiometric dye imaging provide novel insights into functions such as mast cell-nerve signaling in the human gut tissues. Such imaging promises to reveal new ionic or molecular targets to normalize nerve sensitization induced by mast cell hyperactivity or mast cell sensitization by neurogenic inflammatory pathways. These targets include proteinase-activated receptor (PAR) 1 or histamine receptors. In patients, optical imaging in the gut in vivo has the potential to identify neural structures and inflammation in vivo. The latter has some risks and potential of sampling error with a single biopsy. Techniques that image nerve fibers in the retina without the need for contrast agents (optical coherence tomography and full-field optical coherence microscopy) may be applied to study submucous neural plexus. Moreover, the combination of submucosal dissection, use of a fluorescent marker, and endoscopic confocal microscopy provides detailed imaging of myenteric neurons and smooth muscle cells in the muscularis propria. Studies of motility and functional gastrointestinal disorders would be feasible without the need for full-thickness biopsy.

A mediator cocktail released from mucosal biopsies of IBS patients excites human and guinea-pig enteric neurons

Introduction: Irritable bowel syndrome (IBS) is one of the most common reasons for referral to a gastroenterologist. Treatment remains unsatisfactory due to the poorly understood pathophysiology of IBS. IBS symptoms may be linked to altered mediator release from the gut epithelium.

Human mast cell mediator cocktail excites neurons in human and guinea‐pig enteric nervous system

Neuroimmune interactions are an integral part of gut physiology and involved in the pathogenesis of inflammatory and functional bowel disorders. Mast cells and their mediators are important conveyors in the communication from the innate enteric immune system to the enteric nervous system (ENS). However, it is not known whether a mediator cocktail released from activated human mast cells affects neural activity in the ENS. We used the Multi-Site Optical Recording Technique to image single cell activity in guinea-pig and human ENS after application of a mast cell mediator cocktail (MCMC) that was released from isolated human intestinal mucosa mast cells stimulated by IgE-receptor cross-linking. Local application of MCMC onto individual ganglia evoked an excitatory response consisting of action potential discharge. This excitatory response occurred in 31%, 38% or 11% neurons of guinea-pig submucous plexus, human submucous plexus, or guinea-pig myenteric plexus, respectively. Compound action potentials from nerve fibres or fast excitatory synaptic inputs were not affected by MCMC. This study demonstrates immunoneural signalling in the human gut and revealed for the first time that an MCMC released from stimulated human intestinal mast cells induces excitatory actions in the human and guinea-pig ENS.

Human mast cell mediators excite neurones in murine and human enteric nervous system

Neuroimmune interactions are an integral part of gut pyhsiology and involved in the pathogenesis of inflammatory as well as functional bowel disorders. Mast cells and their mediators are pivotal to communicate information from the innate enteric immune system to the enteric nervous system. So far it is not known whether a mediator cocktail released from activated human mast cells affect neural activity and whether mast cell mediators have effects on human enteric nerves. We used the Multi-Site Optical Recording Technique to image single cell activity in guinea-pig and human enteric nervous system after application of a mast cell mediator cocktail (MCMC) that was released from isolated human intestinal mucosa mast cells stimulated by IgE-receptor crosslinking. We studied 106 nerve cells from 8 human submucous plexus preparations, 64 nerve cells from 9 guinea-pig submucous plexus preparations and 1095 nerve cells from 4 guinea-pig myenteric plexus preparations. Local application of MCMC for 500–100ms onto individual ganglia evoked an excitatory response consisting of action potential discharge. This postsynaptic excitatory response occurred in 31%, 38% or 11% neurons of guinea-pig submucous plexus, human submucous plexus, or guinea-pig myenteric plexus, respectively. Fast excitatory synaptic inputs were not affected by MCMC indicating that MCMC had no presynaptic action. This study revealed for the first time that a mast cell mediator cocktail released from stimulated human intestinal mast cells induces excitatory actions in the human and murine enteric nervous system. We thereby present evidence for the concept that immunoneural signals arising from mast cells are able to enhance activity in the enteric nervous system.

Autonomic regulation. i-NANC/e-NANC.

The excitatory and inhibitory nonadrenergic/noncholinergic (e-NANC, i-NANC) systems have been extensively studied. The terms excitatory and inhibitory apply to airway smooth muscle, but the neurotransmitters also act on other targets-blood vessels, glands, the epithelium-where individual actions may be the opposite. Thus, the nomenclature is unsatisfactory. Of the dozen or more putative NANC transmitters, criteria to establish their roles have been met only for vasoactive intestinal polypeptide (VIP), nitric oxide (NO), and substance P/neurokinin A (SP/NKA). VIP and NO co-localize in vagal motor nerves, but they are also found in sympathetic and sensory nerves. In general they have similar actions on target tissues, and their relative importance may vary with species. SP/NKA, released from sensory nerves, is thought to mediate neurogenic inflammation, a process that may include airway smooth muscle contraction, at least in rodents. The evidence for neurogenic inflammation in humans is weak. On the motor side, and also possibly on the sensory, different nerves seem to contain different selections of neurotransmitters, but it is not known if there are different motor controls for these nerves. Cotransmission presents a major conceptual and experimental problem, since the two or more transmitters may give opposite instructions to the target tissue. Inevitably most of the studies on the NANC systems are on isolated rodent tissues, and although quantitative, they indicate little of what happens in vivo, and certainly not in humans. The cocktail of mediators that must be released from nerves and associated cells in airway tissues during pathophysiologic processes may refresh physiologists, but little is known about the concentrations of the ingredients or about the strength of their actions and their interactions on different target tissues in the mucosa.

Rapid detection of bacteria by an amperometric electrode system — a comparison of some redox mediators

The rate of reduction of various mediators and mediator cocktails by E. coli , together with reoxidation currents at an amperometric electrode were examined. When used singly at their optimum concentrations, thionine, ferricyanide and 1,4-benzoquinone gave similar rates of current increase [21.0–25.6 pA min −1 (10 5 cfu) −1 ]. The presence of 1,4-benzoquinone enhanced the rate of reduction of low concentrations (1 mM) of ferricyanide but decreased the reduction rate at high concentrations (28 mM) of ferricyanide. However, the amperometric signal was increased to approximately 42 pA min −1 (10 5 cfu) −1 . Applications of this mediator cocktail to the detection of pure cultures of E. coli allowed the detection of approximately 10 −6 cfu ml −1 . However, when the method was applied to meat samples, bacteria were not detected due to the high level of background current.